JP5351770B2 - Low molecular weight heparins comprising at least one covalent bond with biotin or a biotin derivative, methods for their production and their use. - Google Patents

Abstract

Heparin derivatives (I) comprising a low molecular weight heparin chain covalently linked to biotin are new. Independent claims are also included for: (1) a process for preparing (I); (2) use of avidin or streptavidin for neutralizing (I). - ACTIVITY : Anticoagulant; Cerebroprotective; Neuroprotective. - MECHANISM OF ACTION : Factor Xa inhibitor; Factor IIa inhibitor.

Description

  The present invention relates to a low molecular weight heparin, more generally a mixture of heparinoid polysaccharides, comprising at least one covalent bond with biotin or a biotin derivative, and further to methods for their preparation, pharmaceutical compositions comprising them and Also related to the therapeutic use of.

  Heparin is a mixture of sulfated mucopolysaccharides of animal origin, has a molecular weight of approximately 15000 daltons (Da) and is used especially for its anticoagulant and antithrombogenic properties. However, heparin has the disadvantage that the conditions of use are limited. Specifically, its high anticoagulant activity (especially its high anti-factor IIa activity) can cause bleeding (Seminars in Thrombosis and Hemostasis, vol. 5, sup. 3, 1999).

  Low molecular weight heparins, particularly between 3000 and 7000 Da, more particularly between 3500 and 5500 Daltons, such as enoxaparin, which are obtained by basic depolymerization of heparin esters and are currently commercially available, are also high Has factor IIa activity.

  Heparin derivatives are known for these undesirable bleeding side effects. However, in the field of thrombus treatment with the above products, the aim is to restore or maintain blood fluidity while avoiding the induction of bleeding. In fact, it is well known that bleeding can be induced in a patient under treatment for some unexpected reason. Surgery may be required for patients undergoing antithrombotic treatment. In addition, during certain surgical procedures, high doses of anticoagulants may be used to prevent blood clotting and it is useful to be able to neutralize them at the end of the surgery. Therefore, there is a need for a neutralizable antithrombotic agent to stop anticoagulant activity at any time.

  Neutralizable antithrombotic agents such as biotinylated synthetic polysaccharides are described in patent applications WO02 / 24754 and WO06 / 030104. Their synthesis carried out on the protected equivalents of these polysaccharides, not on the polysaccharides themselves, especially including grafting biotin or biotin derivatives, is not applicable to the compounds of the invention. The reason for this is a mixture of heparin-based polysaccharides, so it is desirable to perform biotinylation on the end product, which is a heterogeneous product, and the grafting of biotin on the end product described in the above-mentioned patent application is not This means that sufficient regioselectivity of the graft position cannot be induced, and the functionalized polysaccharide chains of low molecular weight heparin cannot all be biotinylated.

  Osmond et al.'S team described several techniques for biotinylation of porcine heparin in Analytical Biochemistry, 31 (2002) pp. 199-207, one of which is reductive amination followed by biotin. It is described as involving the coupling of biotin at the reducing end of heparin via coupling. However, under the operating conditions described in the literature, biotinylated heparin cannot be obtained completely and reproducibly, and these operating conditions are due to the structural diversity of heparin and the polysaccharide chain present in commercially available heparin. The actual structure is not taken into account. The latter heparin contains most of the polysaccharide chain containing a degraded glycoserine at the reducing end, which cannot be functionalized with biotin according to the protocol described by Osmond et al. Therefore, the operating conditions for biotinylation of porcine heparin described in the above publication are completely or regenerated while having the expected properties, such as sufficient degree of biotinylation to allow effective neutralization. Heparin cannot be biotinylated as possible.

  The team of Tseng et al., Biomaterials, 27 (2006), pages 2627-2636, describes a technique for functionalizing heparin with biotin and then immobilizing heparin on the film by interaction with avidin. . Biotinylation of heparin is performed via oxidation with iodine, followed by formation of a lactone, followed by coupling with a biotin 2- (4-aminophenyl) ethylamine derivative. However, the operating conditions presented by Tseng et al. Do not presuppose complete and reproducible production of biotinylated heparin at the reducing end, in particular the oxidation step can be selective with respect to the reducing end. Or heparin's biological activity is not shown to be preserved after such treatment.

International Publication No. 02/24754 Pamphlet International Publication No. 06/030104 Pamphlet

Seminars in Thrombosis and Hemostasis, vol. 5, sup. 3, 1999 Osmond et al., Analytical Biochemistry, 31 (2002) 199-207. Tseng et al., Biomaterials, 27 (2006), 2627-2636.

  Accordingly, Applicants have identified novel low molecular weight heparins that can be neutralized by avidin or streptavidin and have biological properties comparable to the starting low molecular weight heparin, particularly anti-factor Xa activity and anti-factor IIa activity. Set for the purpose of providing itself.

  The present invention relates to novel modified low molecular weight heparins, hereinafter referred to as “biotinylated low molecular weight heparins”, which have an average molecular weight between 3000 and 7000 Da, these constituent polysaccharides being It is characterized in that it is covalently bound to biotin or a biotin derivative at the reducing end.

  Surprisingly, the introduction of biotin or a biotin derivative at the reducing end of the polysaccharide chain does not modify the pharmacological activity of low molecular weight heparin. In particular, the novel biotinylated low molecular weight heparin that is the object of the present invention has antithrombotic activity comparable to native low molecular weight heparin, ie, heparin before biotinylation.

  The novel biotinylated low molecular weight heparin has significant advantages over native low molecular weight heparin, and in an emergency, the novel biotinylated low molecular weight heparin can be quickly neutralized using a specific antidote. obtain. This particular antidote is a tetramer or monomeric avidin or streptavidin having a mass equal to about 66000, 16400 and 60000 Da, respectively (The Merck Index, 12th edition, 1996, MN. 920, 151-152; revised Pierce Avidin-Biotin Handbook).

  The novel biotinylated low molecular weight heparin further has a useful advantage in therapeutic indications that at the same time the dose used is increased, the risk of bleeding is reduced, thus the novel biotinylated Low molecular weight heparin may be useful in the art of arterial therapy.

  In the context of the present invention, the term “low molecular weight heparin” means a mixture of sulfated polysaccharides having the general structure of heparin's constituent polysaccharides, which have an average molecular weight of 3000 to 7000 Da, Obtained by depolymerization. Hereinafter, the term “low molecular weight heparin” or “native low molecular weight heparin” refers to a mixture of polysaccharides prior to biotinylation, whereas the term “biotinylated low molecular weight heparin” refers to biotin or Represents a compound according to the invention comprising a covalent bond with a derivative of

  The term “reducing end” refers to a terminal glucosamine or mannosamine (mannosamine resulting from epimerization of glucosamine in a basic medium) according to the following formula (II):

［式中、
−Ｘは、ＨまたはＳＯ_３Ｎａを表し、
−Ｙは、ＣＯＣＨ_３またはＳＯ_３Ｎａを表し、
−波線は、これが結合しているピラノース環平面の下または上のいずれかに位置する結合を表す（下：グルコサミン、上：マンノサミン）。］
に対応する、環状ヘミアセタール官能基を有する多糖類鎖の末端を表す。

[Where:
-X represents H or _SO 3 Na,
-Y represents COCH ₃ or _SO 3 Na,
-The wavy line represents a bond located either below or above the plane of the pyranose ring to which it is attached (bottom: glucosamine, top: mannosamine). ]
The terminal of the polysaccharide chain which has a cyclic hemiacetal functional group corresponding to is represented.

  Among the low molecular weight heparins that can be used in the present invention, some of these may appear to have at least 75% of these polysaccharide chains contain a hemiacetal-type glucosamine at their reducing end, A functionalizable polysaccharide. Certain polysaccharide chains present in the mixture may be in 1,6-anhydro form up to a content of 25% or less, and such polysaccharides cannot be functionalized with biotin or biotin derivatives.

  The term “functionalizable constituent polysaccharide” of a mixture means a polysaccharide containing a hemiacetal-type glucosamine as defined by formula (II) at the reducing end.

  The term “heparin constituent polysaccharide” means a polysaccharide characterized by repeating disaccharide units containing a uronic acid residue (D-glucolonic acid or L-iduronic acid) and a D-glucosamine residue, D -Glucosamine may be N-sulfated or N-acetylated. The disaccharide unit may also be O-sulfated at the C6 and / or C3 position of D-glucosamine and at the C2 position of uronic acid (Heparin-binding proteins, H. Edward Conrad, 1998, Page 1).

  The biotinylated low molecular weight heparin according to the present invention has these constituent polysaccharides represented by the formula (I):

［式中、
−ｉは０または１に等しく、
−Ｒ１は、式（ａ）または（ｂ）：

[Where:
-I is equal to 0 or 1,
-R1 is represented by the formula (a) or (b):

｛式中、ｊおよびｋは、同一でありまたは異なってよく、１から１０の任意の値をとり得る整数である。｝の連鎖を表し、
−Ｂｉｏｔは、ビオチン基またはビオチン誘導体を表し、
−ＰＥは、ヘパリンの構成多糖類の一般式を有する多糖類鎖を表し、
−ＸはＨまたはＳＯ_３Ｎａを表し、
−ＹはＳＯ_３ＮａまたはＣＯＣＨ_３を表し、
−波線は、これが結合しているピラノース環平面の下または上のいずれかに位置する結合を表す。］
およびこれらの薬学的に許容される塩、
に対応することを有利に特徴とする。

{Wherein j and k are the same or different and are integers that can take any value from 1 to 10. } Chain,
-Biot represents a biotin group or biotin derivative,
-PE represents a polysaccharide chain having the general formula of the constituent polysaccharide of heparin;
-X represents H or _SO 3 Na,
-Y represents _SO 3 Na or COCH _3,
The wavy line represents a bond located either below or above the pyranose ring plane to which it is attached. ]
And pharmaceutically acceptable salts thereof,
It is advantageous to correspond to

  The above-described biotin (Biot) group is a group derived from hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-4-pentanoic acid. Advantageously, the Biot group of general formula (I) according to the invention has the formula (c):

に対応する。

Corresponding to

  Biotin derivatives are commercially available ("Pierce" biotin-avidin product catalog, 2005, pages 7-11) or can be prepared using standard methods known to those skilled in the art. In particular, mention may be made of the biotin derivatives described in patent application WO 02/24754.

  In the biotinylated low molecular weight heparin according to the invention, the subscript i may be equal to 0, in which case the binding to biotin or a biotin derivative is an amine function carried by the monosaccharide unit at the reducing end of the polysaccharide chain. Done directly on the basis.

Alternatively, i may be equal to 1, and the bond with the biotin group or biotin derivative may consist of, for example, the chain of formula (a) above, where j is equal to 5 or Wherein j and k are the same and are equal to 5. Therefore, in the above formula (I), R1 is, for example, a chain of the formula —CO— (CH ₂ ) ₅ —NH or —CO— (CH ₂ ) ₅ —NH—CO— (CH ₂ ) ₅ —NH—. Can be represented.

  Biotinylated low molecular weight heparin according to the present invention has at least 60%, preferably at least 80%, even more preferably at least 90% of these constituent polysaccharides shared with biotin or a biotin derivative at the reducing end. It has a bond.

  The low molecular weight heparin used in the present invention may be selected from, for example, enoxaparin, ardeparin, bemiparin, parnaparin and tinzaparin.

As defined in patents US5389618 and USRE38743, the low molecular weight heparins used in the present invention are in particular:
-9% to 20% of these constituent polysaccharides have an average molecular weight of less than 2000 Da;
-5% to 20% of these constituent polysaccharides have an average molecular weight above 8000 Da;
-60% to 86% of these constituent polysaccharides have an average molecular weight between 2000 and 8000 Da;
The ratio between the weight average molecular weight and the number average molecular weight is between 1.3 and 1.6;
The low molecular weight heparin has better bioavailability and antithrombotic activity than heparin and has an average molecular weight between about 3500 and 5500 Da;
Can be a thing.

  The present invention is also directed to biotinylated low molecular weight heparin in the form of any of these pharmaceutically acceptable salts.

The object of the present invention is further
a) performing a reductive amination on the low molecular weight heparin defined above in the presence of an amine salt and a reducing agent at a temperature between 20 and 80 ° C .;
b) Then using the activating group-(R1) _i -Biot-, where R1, i and Biot are as defined for formula (I) above, in an aqueous or organic medium Carrying out the acylation in the presence of a base;
A method for preparing the biotinylated low molecular weight heparin described above.

  The steps of the above preparation methods are carried out by analytical HPLC monitoring, in particular by the SAX type, for example using the method described in patent application WO 2004/027087 or optionally via LC-MS, for example Robert J . Linhardt, J .; Biol. Chem. 2004, 279 (4), pages 2608-2615.

  Biotinylated low molecular weight heparin can be analyzed and characterized according to analytical conditions described by the supplier by affinity chromatography on supported monomeric avidin, sold by Pierce.

After reductive amination step a), it is particularly confirmed that at least 90% of the low molecular weight heparin constitutive polysaccharides carry a —NH ₂ functional group at their reducing end (amino reduction). Polysaccharides).

  It has been particularly confirmed that after acylation step b) at least 90% of the amino-reduced polysaccharide is biotinylated.

  The overall yield of the process for preparing biotinylated low molecular weight heparin according to the invention is therefore at least 80%, preferably at least 90%.

  The process for preparing compounds according to the present invention uses low molecular weight heparin prepared as previously reported in the literature as the starting low molecular weight heparin ("native" low molecular weight heparin). In particular, see patent US RE38743 for enoxaparin, US4757057 for ardeparin, EP0293539 for bemiparin, US4791195 for parnaparin and US5106734 for tinzaparin.

In step a) the reductive amination of the above methods of preparing the amine salt may be a tetramer amine salts, a halogen of the formula NH 4 _{Z (where,} Z is chlorine, fluorine, bromine or iodine atom Advantageously, it is an ammonium halide salt corresponding to (represents an atom).

  In step a) of the reductive amination of the above preparation method, the reducing agent may be a borohydride salt, such as a cyanoborohydride salt.

  In step a) of the reductive amination of the above preparation process, the temperature is advantageously between 50 and 80 ° C.

  In the acylation step b) of the above preparation method, the base may be in the form of a carbonate or bicarbonate, in particular a sodium or potassium salt, or any water-soluble or organic-soluble salt known to those skilled in the art. It can be an organic base.

  In the acylation step b) of the above preparation process, the term “organic medium” means, for example, dichloromethane or dimethylformamide.

The preparation method of biotinylated low molecular weight heparin according to the present invention comprises the following steps:
a) performing reductive amination of low molecular weight heparin at a temperature between 50 and 80 ° C. in the presence of ammonium halide salt and borohydride salt;
b) then performing the acylation using the activated ester form of the-(R1) _i -Biot group defined above in the presence of a base in an aqueous medium;
Are advantageously included.

The biotinylated derivative- (R1) _i -Biot defined above can be used directly in the acylation reaction in the form of an activated ester and performed in situ using standard coupling conditions known to those skilled in the art. Or can be generated. In particular, activated ertel in the form of N-hydroxysuccinimide derivatives or 3-sulfo-N-hydroxysuccinimide derivatives can be used.

  The preparation method according to the present invention is illustrated in Scheme 1.

  According to Scheme 1, low molecular weight heparin is subjected to reductive amination, and derivative A containing a free amino function at the reducing end is produced in the presence of a reducing agent such as amine salts and borohydride salts. The

This derivative can then be acylated in the presence of a base to provide biotinylated derivative B via reaction with the activated biotin derivative defined above,-(R1) _i -Biot. For example, this reaction may be performed by using 3-sulfosuccinimidyl 6-biotinamidohexanoyl which is an ester when R1 represents a chain of —CO— (CH ₂ ) ₅ —NH—CO— (CH ₂ ) ₅ —NH. When the sodium salt of hexanoate is used and R1 represents a chain of —CO— (CH ₂ ) ₅ —NH—, the sodium salt of 3-sulfosuccinimidyl 6-biotinamidohexanoate, which is an ester, is used. Or when R1 is not present (i = 0), can be carried out using the sodium salt of biotinoyl-3-sulfosuccinimidyl ester.

  It should be understood that in Scheme 1, derivatives A and B are theoretical representations because they are actually low molecular weight heparins of a mixture of polysaccharide chains.

  Hereinafter, examples of synthesizing biotinylated low molecular weight heparins according to the present invention and examples of synthesizing various intermediates useful for obtaining them will be described in detail below.

The following abbreviations are used:
HPLC: high performance liquid chromatography,
SAX: strong anion exchange chromatography,
LC-MS: liquid chromatography-mass spectrometry,
qs: sufficient amount,
LC: long chain, LC-LC corresponding to 6-aminohexanoyl chain: represents two LC chains, corresponding to amidohexanoyl-6-aminohexanoyl chain,
Sulfo-NHS: sodium salt of 3-sulfosuccinimidyl ester,
Heparinase 1: Heparin lyase I enzyme (EC 4.2.2.7) from Flavobacterium heparinum

2 is a diagram illustrating reaction monitoring of enoxaparin conversion by HPLC SAX according to Example 1. FIG. It is the figure which illustrated the analysis by HPLC SAX of the biotinylated fraction and non-biotinylated fraction obtained after letting the product obtained according to Example 1 pass through the column which supported the avidin monomer. It is the figure which illustrated the analysis by HPLC SAX of the biotinylated fraction obtained after letting the product obtained according to Example 2 pass through the column which supported the avidin monomer, and a non-biotinylated fraction. It is the figure which illustrated the analysis by HPLC SAX of the biotinylated fraction obtained after passing the product obtained according to Example 3 through the column which supported the avidin monomer, and a non-biotinylated fraction.

NH LC biotinoyl enoxaparin Enoxaparin is a low molecular weight heparin obtained according to the method described in patent US RE38743. This is converted to a biotinylated derivative according to the reaction sequence in Scheme 2. That is, enoxaparin is converted through reductive amination reaction to compound 1 having an amino functional group at the reducing end, which is then converted to sodium 3-sulfosuccinimidyl 6-biotinamidohexanoate. It is converted to biotinylated compound 2 via reaction with a salt.

1.1: 1-Aminoenoxaparin 1 g of enoxaparin is dissolved in 40 ml of 5M aqueous ammonium chloride solution. 1 g of sodium cyanoborohydride is added to the resulting solution. The mixture is maintained at 60 ° C. for 24 hours. The solution is cooled to a temperature of about 20 ° C. and diluted with water (qs 100 ml). The resulting filtrate is desalted with a Sephadex G10 column and then lyophilized. 824 mg of white lyophilizate is obtained. The actual yield is 82%. The product is examined by HPLC SAX (see FIG. 1) and used for the biotinylation step without further purification.

1.2: NH LC Biotinyl enoxaparin 200 mg of 1-aminoenoxaparin is dissolved in 5 ml of 0.5 M sodium bicarbonate solution at a temperature of about 20 ° C. 136 mg of sulfo-NHS-LC-biotin is added to the resulting solution. The solution is stirred for 1 hour at a temperature of about 20 ° C. The resulting suspension is diluted with 10 ml of 0.5 M sodium bicarbonate solution. 136 mg of sulfo-NHS-LC-biotin are added and the resulting mixture is stirred for 18 hours. An additional 136 mg of sulfo-NHS-LC-biotin is added and the reaction mixture is stirred for 1 hour. An additional 70 mg of sulfo-NHS-LC-biotin is added and the reaction mixture is stirred for 3 hours. The reaction medium obtained is diluted with water (qs 200 ml), filtered through a 0.45 μm membrane and then desalted on a Sephadex G10 column. The obtained fraction is injected into a Q-Sepharose column. The product is eluted with a gradient of water and then sodium perchlorate. The collected fractions are desalted on a Sephadex G10 column. The resulting product is purified again by passing through a Q-Sepharose column and desalted with Sephadex G10. The final fraction collected is lyophilized. 190 mg of white lyophilizate is obtained. The actual yield is 87%.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 1.3-1.8 (12H, m), 2.05 (CH ₃ CO, s). 25 _(2CH 2 CO biotin, m), 2.80 (1H, d, 12Hz), 3.03 (1H, dd, 12 and 5Hz), 3.15-5.65 (polysaccharides protons), 5.99 (1H, d, 4 Hz).

  The resulting product is examined by HPLC SAX. FIG. 1 (FIG. 1/4) illustrates reaction monitoring by HPLC SAX that enoxaparin has been converted to derivative 1 having an amino functional group at the reducing end via a reductive amination reaction (Scheme 2). Please refer.) This derivative is then converted to biotinylated derivative 2 via reaction with the sodium salt of 3-sulfosuccinimidyl 6-biotinamidohexanoate. The analysis method used is described in the patent application WO 2004/027087. FIG. 1 shows that a species containing a functionalizable glucosamine was converted to a derivative containing an amino functional group at the reducing end with a degree of conversion greater than 90% to give 1-aminoenoxaparin. FIG. 1 also shows that species containing an amino functional group at the reducing end can be biotinylated with a conversion greater than 90% via reaction with the sodium salt of 3-sulfosuccinimidyl 6-biotinamidohexanoate. It is also shown that it was converted to a derivative and NH LC biotinyl enoxaparin was obtained.

  As an example, FIG. 1 shows the peaks corresponding to the main compound present in the oligosaccharide mixture obtained according to Example 1, the structure of which is represented below (the nomenclature used is the patent application The same as the nomenclature of the 2004/027087 pamphlet).

LC-MS analysis can confirm the structure of these compounds via mass spectrometry corresponding to the acidic form of the product: Δlsls _ld m / z = 1154; Δlsls _ld ls _ld m / z = 1733; _{Δlsls ld ls ld ls ld m /} z = 2308; Δlsls ld1,6-anhydro m / z = 1056; Δlsls ld ls ld1,6-anhydro m / z = 1633; Δlsls ld ls ld ls ld1,6-anhydro m / Δlsls _ld NH ₂ m / z = 1155; Δlsls _ld ls _ld NH ₂ m / z = 1732; Δlsls _ld ls _ld ls _ld NH ₂ m / z = 2309; Δ lsls _ld NH LC z Biot 1494; Δlsls _ld ls _ld NH LC Biot m / z = 2071; Δ lsls _ld ls _{ld ld ld ld} NH LC Biot m / z = 2648.

  Furthermore, the product obtained according to Example 1 can be injected into a column supporting an avidin monomer. Elution is performed according to the conditions described by the supplier's Pierce. The biotinylated fraction (with affinity for avidin) and the non-biotinylated fraction (without affinity for avidin) thus obtained are then injected into HPLC SAX (FIG. 2, drawing). See 2/4.) FIG. 2 illustrates the analysis by HPLC SAX of the biotinylated and non-biotinylated fractions obtained after passing through a column supporting an avidin monomer. FIG. 2 shows that species containing functionalizable glucosamine have been converted to the corresponding biotinylated species with a degree of conversion greater than 90%. The fraction with no affinity is mainly composed of 1,6-anhydro derivatives, which in nature cannot be converted into biotinylated derivatives. Some structures of the main peaks are given as examples characterizing the resulting product (see structure illustrated above).

NH Biotinoyl Enoxaparin Enoxaparin, a low molecular weight heparin obtained according to the method described in US Reissue Pat. No. 38743, is converted to a biotinylated derivative according to the reaction sequence described in Scheme 3. That is, enoxaparin is converted via reductive amination reaction to compound 1 containing an amino functional group at its reducing end, after which this derivative is reacted with the sodium salt of biotinoyl-3-sulfosuccinimidyl ester. To biotinylated compound 3.

  200 mg of 1-aminoenoxaparin is dissolved in 5 ml of 0.5 M sodium bicarbonate solution at a temperature of about 20 ° C. 107 mg of sulfo-NHS-biotin is added to the resulting solution. The solution is stirred at a temperature of about 20 ° C. for 1 hour 30 minutes. The resulting suspension is diluted with 10 ml of 0.5 M sodium bicarbonate solution. 107 mg of sulfo-NHS-biotin are added and the resulting mixture is stirred for 3 hours. The resulting reaction medium is diluted with water (qs 150 ml), filtered through a 0.45 μm membrane and then injected onto a Q-Sepharose column. The product is eluted with a gradient of water and then sodium perchlorate. The collected fractions are desalted on a Sephadex G10 column. The collected fractions are lyophilized. 190 mg of white lyophilizate is obtained. The actual yield is 90%.

The resulting product was examined by HPLC SAX (see FIG. 3, FIG. 3/4 “comprehensive” graph) and the species containing the amino functionality at the reducing end was biotinol-3-sulfosuccinimid. It has been confirmed that it has been converted to a biotinylated derivative with a degree of conversion of more than 90% through reaction with the sodium salt of luster.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 1.4-1.8 (6H, m), 2.05 (CH ₃ CO, s). 3 (CH ₂ CO biotin, m), 2.80 (1H, dd, 12 and 7 Hz), 3.03 (1H, m), 3.20-5.65 (polysaccharide proton), 5.98 (1H , D, 4 Hz).

  The product obtained according to Example 2 is injected into a column supporting an avidin monomer. Elution is performed according to the conditions described by the supplier's Pierce. The resulting biotinylated fraction (having affinity for avidin) and non-biotinylated fraction (no affinity for avidin) are then injected into HPLC SAX (see Figure 3, Figure 3/4) I want to be.) The fraction with no affinity is mainly composed of 1,6-anhydro derivatives, which in nature cannot be converted into biotinylated derivatives.

  As an example, FIG. 3 describes the structure of certain major compounds of an oligosaccharide mixture. The referenced structure is shown below.

LC-MS analysis can confirm the structure of these compounds via mass spectrometry corresponding to the acidic form product: Δlsls _{ld1,6-anhydro} m / z = 1056; Δlsls _ld ls _{ldl, 6 -anhydro m / z = 1633; Δlsls} ld ls ld ls ldl, 6-anhydro m / z = 2210; Δlsls ld NH Biot m / z = 1381; Δlsls ld ls ld NH Biot m / z = 1958; Δlsls ld ls ld ls _ld NH Biot m / z = 2535.

NH-LC-LC Biotinoyl Enoxaparin, a low molecular weight heparin obtained according to the method described in US Reissue Pat. No. 38743, is converted to a biotinylated derivative according to the reaction sequence described in Scheme 4. Is done. That is, enoxaparin is converted through reductive amination to compound 1 containing an amino functional group at the reducing end, after which this derivative is converted to the ester 3-sulfosuccinimidyl 6-biotinamidohexanoylhexa Conversion to biotinylated compound 4 by reaction with sodium salt of noate.

  200 mg of 1-aminoenoxaparin is dissolved in 5 ml of 0.5 M sodium bicarbonate solution at a temperature of about 20 ° C. 164 mg of sulfo-NHS-LC-LC-biotin is added to the resulting solution. The solution is stirred at a temperature of about 20 ° C. for 2 hours. The suspension is diluted with 10 ml of 0.5 M sodium bicarbonate solution. 164 mg of sulfo-NHS-LC-LC-biotin is added and the resulting mixture is stirred for 5 hours. The reaction medium obtained is diluted with water (qs 150 ml), filtered through a 0.45 μm membrane and then injected onto a Q-Sepharose column. The product is eluted with a gradient of water and then sodium perchlorate. The collected fractions are desalted on a Sephadex G10 column. The collected fractions are lyophilized. 210 mg of a white lyophilizate is obtained. The actual yield is 92%.

The resulting product was examined by HPLC SAX (see FIG. 4, FIG. 4/4 “Comprehensive” graph), and the species containing the amino functionality at the reducing end was 3-sulfosuccinimidyl 6 -It has been confirmed that it has been converted to a biotinylated derivative with a degree of conversion greater than 90% via reaction with the sodium salt of biotinamidohexanoylhexanoate.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 1.3-1.8 (16H, m), 2.05 (CH ₃ CO, s). 25 (6H, m), 2.80 (1 H, dd, 12 and 7 Hz), 3.03 (1 H, m), 3.20-5.65 (polysaccharide proton), 5.98 (1 H, d, 4 Hz).

  The product obtained according to Example 3 is injected into a column supporting an avidin monomer. Elution is performed according to the conditions described by the supplier's Pierce. The resulting biotinylated fraction (having affinity for avidin) and non-biotinylated fraction (no affinity for avidin) are then injected into HPLC SAX (see FIG. 4). The fraction with no affinity is mainly composed of 1,6-anhydro derivatives, which in nature cannot be converted into biotinylated derivatives.

  The structure of the main compound is confirmed by LC-MS coupling.

  As an example, FIG. 4 describes the structure of certain major compounds of an oligosaccharide mixture. The referenced structure is shown below.

By LC-MS analysis, the structure of the above compound can be confirmed via mass spectrometry corresponding to the acidic form product: Δlsls _{ld1,6-anhydro} m / z = 1105; Δlsls _ld ls _{ld ld6 -Anhydro} m / z = 1633; Δlsls _ld ls _ld ls _{ld1,6-anhydro} m / z = 2210;
Δlsls _ld NH LC LC Biot m / z = 1607; Δlsls _ld ls _ld NH LC LC Biot m / z = 2184; Δlsls _ld ls _{ld ld ld} NH LC LC Biot m / z = 2761.

Tin-zaparin, a low molecular weight heparin of about 6000 daltons, obtained by treatment with NH-LC biotinoyltin zaparin heparinase 1, can also be converted to a biotinylated derivative according to the reaction sequence described in Scheme 5. That is, tinzaparin is converted via reductive amination reaction to compound 5 containing an amino functional group at the reducing end, after which this derivative is sodium 3-sulfosuccinimidyl 6-biotinamidohexanoate. It is converted to biotinylated compound 6 through reaction with a salt.

4.1: 1-aminotinzaparin 250 mg of tinzaparin is dissolved in 10 ml of 5M aqueous ammonium chloride solution. 250 mg sodium cyanoborohydride is added to the resulting solution. The mixture is maintained at 70 ° C. for 20 hours. The solution is cooled to a temperature of about 20 ° C. and diluted with water (qs 20 ml). The resulting filtrate is desalted with a Sephadex G10 column and then lyophilized. 215 mg of a white lyophilizate is obtained. The actual yield is 86%.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 2.05 (CH ₃ CO, s), 3.10 and 3.40 (each 1 H, m, CH ₂ NH _2), 3.20-5.65 (polysaccharides protons), 5.98 (1H, d, 4Hz).

  Compounds can be examined by HPLC SAX (see FIG. 1) using the method outlined above in Example 1.

  The product is used for the biotinylation step without further purification.

4.2: NH LC Biotinoyltinzaparin 100 mg of 1-aminotinzaparin is dissolved in 2.5 ml of 0.5 M sodium bicarbonate solution at a temperature of about 20 ° C. 47 mg of sulfo-NHS-LC-biotin is added to the resulting solution. The solution is stirred at a temperature of about 20 ° C. for 1 hour 45 minutes. The resulting suspension is diluted with 5 ml of 0.5 M sodium bicarbonate solution. 47 mg of sulfo-NHS-LC-biotin is added and the resulting mixture is stirred for 6 hours. An additional 47 mg of sulfo-NHS-LC-biotin is added and the reaction mixture is stirred for 20 hours. The suspension is diluted again with 1 ml of 0.5 M sodium bicarbonate solution and an additional 47 mg of sulfo-NHS-LC-biotin is added. The reaction mixture is stirred for 20 hours. The suspension is re-diluted with 6.5 ml of 0.5 M sodium bicarbonate solution and an additional 47 mg of sulfo-NHS-LC-biotin is added. The reaction mixture is stirred for 22 hours, then diluted with water (qs 100 ml), filtered through a 0.45 μm membrane and injected onto a Q-Sepharose column. The product is eluted with a gradient of water and then sodium perchlorate. The collected fractions are desalted on a Sephadex G10 column. The final fraction collected is lyophilized. 110 mg of a white lyophilizate is obtained. The actual yield is quantitative.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 1.3-1.8 (12H, m), 2.05 (CH ₃ CO, s). 25 (4H, m), 2.80 (1 H, dd, 12 and 7 Hz), 3.03 (1 H, m), 3.20-5.65 (polysaccharide proton), 5.98 (1 H, d, 4 Hz).

  Compound 1-aminotinzaparin and the resulting NH LC biotinoltinzaparin can also be characterized via the HPLC SAX method previously used in Example 1. This HPLC test shows that a species containing a functionalizable glucosamine is converted to a derivative containing an amino functional group at the reducing end with a degree of conversion greater than 90%, resulting in 1-aminotinzaparin. Yes. Furthermore, this HPLC test showed that species containing an amino functional group at the reducing end, with a conversion of more than 90%, via reaction with the sodium salt of 3-sulfosuccinimidyl 6-biotinamidohexanoate, It has also been shown to be converted to a biotinylated derivative to give NH LC biotinoltinzaparin.

  In the same manner as in Example 1, the structure of the main compound can be confirmed by LC-MS analysis.

  The resulting product can be further injected into a column supporting an avidin monomer. Elution is performed according to the conditions described by the supplier's Pierce. The resulting biotinylated fraction (having affinity for avidin) and non-biotinylated fraction (no affinity for avidin) can be examined by HPLC SAX.

NH LC biotinoyl bemiparin, a low molecular weight heparin of about 3500 daltons obtained via alkaline depolymerization, bemiparin can also be converted to a biotinylated derivative according to the reaction sequence described in Scheme 6 below. That is, bemiparin is converted via reductive amination to compound 7 containing an amino functional group at the reducing end, after which this derivative is sodium 3-sulfosuccinimidyl 6-biotinamidohexanoate. It is converted to biotinylated compound 8 via reaction with a salt.

5.1: 1-aminobemiparin:
250 mg bemiparin is dissolved in 10 ml 5M aqueous ammonium chloride solution. 250 mg sodium cyanoborohydride is added to the resulting solution. The mixture is maintained at 70 ° C. for 20 hours. The solution is cooled to a temperature of about 20 ° C. and diluted with water (qs 20 ml). The resulting solution is desalted on a Sephadex G10 column and then lyophilized. 227 mg of a white lyophilizate is obtained. The actual yield is 91%.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 2.05 (CH ₃ CO, s), 3.10 and 3.40 (each 1 H, m, CH ₂ NH _2), 3.20-5.80 (polysaccharides protons), 5.98 (1H, d, 4Hz).

  The compounds can be examined by HPLC SAX using the method outlined above in Example 1.

  The resulting product is used for the biotinylation step without further purification.

5.2: NH LC biotinoyl bemiparin:
100 mg of 1-aminobemiparin is dissolved in 5 ml of 0.5 M sodium bicarbonate solution at a temperature of about 20 ° C. 80 mg of sulfo-NHS-LC-biotin is added to the resulting solution. The solution is stirred at a temperature of about 20 ° C. for 2 hours. The resulting suspension is diluted with 10 ml of 0.5 M sodium bicarbonate solution. 80 mg of sulfo-NHS-LC-biotin is added and the resulting mixture is stirred for 2 hours. An additional 40 mg of sulfo-NHS-LC-biotin is added and the reaction mixture is stirred for 20 hours. The reaction medium obtained is diluted with water (qs 50 ml) and then desalted on a Sephadex G10 column. The obtained fraction is injected into a Q-Sepharose column. The product is eluted with a gradient of water and then sodium perchlorate. The collected fractions are desalted on a Sephadex G10 column. The resulting product is purified again through a Q-Sepharose column and desalted on a Sephadex G10 column. The final fraction collected is lyophilized. 101 mg of a white lyophilizate is obtained. The actual yield is 92%.
¹ H NMR spectrum of oligosaccharide mixture in D ₂ O (25 ° C., δ (ppm)): 1.3-1.8 (12H, m), 2.05 (CH ₃ CO, s). 25 (4H, m), 2.80 (1 H, dd, 12 and 7 Hz), 3.03 (1 H, m), 3.20-5.65 (polysaccharide proton), 5.98 (1 H, d, 4 Hz).

  Compound 1-aminobemiparin and the resulting NH LC biotinoyl bemiparin can also be characterized via the HPLC SAX method previously used in Example 1. This HPLC test shows that species containing functionalizable glucosamine are converted to derivatives containing an amino functional group at their reducing end with a degree of conversion greater than 90%, resulting in 1-aminobemiparin. Furthermore, this HPLC test showed that species containing an amino functional group at the reducing end, with a conversion of more than 90%, via reaction with the sodium salt of 3-sulfosuccinimidyl 6-biotinamidohexanoate, It is also shown that it is converted to a biotinylated derivative to give NH LC biotinyl bemiparin.

  In the same manner as in Example 1, the structure of the main compound can be confirmed by LC-MS analysis.

  The resulting product can be further injected into a column supporting an avidin monomer. Elution is performed according to the conditions described by the supplier's Pierce. The resulting biotinylated fraction (having affinity for avidin) and non-biotinylated fraction (no affinity for avidin) can be examined by HPLC SAX.

  The compounds according to the invention have been subjected to biochemical and pharmacological studies.

1. Measurement of anti-factor IIa and anti-factor Xa activity Anti-factor IIa (anti-FIIa) activity and anti-factor Xa (anti-FXa) activity in human plasma or buffer systems are analyzed via a chromogenic method. That is, anti-factor IIa activity is examined using the Actichrome heparin anti-factor II kit (American diagnostic) containing chromogenic substrate S-2238, α-thrombin and human ATIII (anti-thrombin III). Anti-FXa activity is measured using a Heparin kit (Instrumentation Laboratory) containing ATIII, Factor Xa and chromogenic substrate S-2765 using an automatic coagulation device ACL7000 (Instrumentation Laboratory). Two analyzes are performed according to the manufacturer's instructions.

The following standards are used to establish a standard calibration curve for the measurement of in vitro activity of biotinylated low molecular weight heparin fractions in human plasma and buffer systems:
First standard for low molecular weight heparin (National Institute for Biological Standards and Control, London, UK, approved in 1987, code number 85/600)
Second standard for low molecular weight heparin (National Institute for Biological Standards and Control, London, UK, approved in 1987, code number 01/608, used from June 2006)
-Enoxaparin (Clexane (R), sanofi-avenis, France) is used as an internal standard.

  For the measurement of anti-FIIa activity, 10 μl of sample or international low molecular weight heparin standard was prepared using human plasma or pH 7.4 buffered antithrombin containing 0.05 M Tris HCl, 0.154 M NaCl. : Dilute to 16. 10 μl of this solution is added to a 96 well microtiter plate. The measurement is repeated 3 times (for 3 wells). The microtiter plate is maintained at 37 ° C. with stirring at 300 rpm. 40 μl of thrombin is added to each well and incubated for exactly 2 minutes. Add 40 μl Spectrozyme. After 90 seconds, the reaction is stopped by adding 40 μl of acetic acid. Absorption is measured using SpectraMax 340 (Molecular Devices) at 405 nm.

  For measurement of anti-FXa activity, samples or international low molecular weight heparin standards are diluted in human plasma or a buffer system at pH 7.4 containing 0.05 M Tris HCl, 0.154 M NaCl. Samples containing heparinoids in plasma or buffer are again diluted 1:20 with working buffer containing ATIII and placed in duplicate in the probe rotor. Factor Xa reagent and chromogenic substrate are poured into the indicated container of the automatic coagulator ACL7000.

  Measurement of anti-FXa activity is performed using the “heparin” protocol integrated in the ACL7000 software. During the analysis, 50 μl of sample (diluted with working buffer) is mixed with 50 μl of factor Xa reagent. After incubation for 60 seconds at 37 ° C., 50 μl of chromogenic substrate at a concentration of 1.1 mM is added and the change in absorption as a function of time is measured at a wavelength of 405 nm.

  The results obtained are described in particular in Table 1.

In this table, MM represents the average molar mass (in daltons), and “correcting” activity can correct for volume dilution effects in the measurement. The corrected activity is calculated as follows:
Correcting activity = (measured activity × MM of prepared compound) / MM of starting material,
Where
MM of the prepared compound: theoretical average molar mass of the prepared compound,
Starting material MM: average molar mass of starting low molecular weight heparin.

  These results indicate that the biotinylated low molecular weight heparin according to the present invention preserves anti-factor X and anti-factor IIa activity comparable to native low molecular weight heparin. Thus, preserving these biological properties enables biotinylated low molecular weight heparin according to the present invention to be used therapeutically.

2. Measurement of anti-FXa activity after neutralization with avidin Neutralization of the effect of biotinylated product in solution with avidin Product-dependent anti-FXa or anti-thrombin activity of FIIa is associated with this activity To measure the effect of avidin binding to biotin, it is measured in the presence of increasing concentrations of avidin.

  The test product is dissolved at 1 mg / ml in water containing 0.9% NaCl. Thereafter, the product was purified from the activity of Factor Xa (Factor Xa, Chromogenix Milan, Italy) or Factor IIa (Factor IIa, laboratory duo sang [Blood Laboratory], Strasbourg) Dilute to obtain a product concentration that can be inhibited in the presence of thrombin, Milan, Italy. This inhibition was then reduced to decreasing concentrations of avidin (Sigma, avidin from egg white, Ref. A-9275, diluted in NaCl): 300, 30, 3, 0.3, 0.03, 0.003, 0 μg / Measure in the presence of ml. Analyze the residual activity of factor Xa (or factor IIa), add specific chromogenic substrate S2222 (Chromogenix, Milan, Italy) for factor Xa and substrate S2238 (Chromogenix, Milan, Italy) for factor IIa To implement.

  Absorbance is read at 405 nm.

Demonstration of Biotinylated Product Binding to Avidin Bound to Beads in Buffer To evaluate the product's ability to bind avidin, the product is placed in contact with the avidin bound to the beads. After centrifuging the mixture, anti-FXa or anti-FIIa activity is measured in the supernatant. This activity can determine the concentration remaining in the medium and thus the percentage of product trapped in the pellet after the mixture has been centrifuged.

  The test product is dissolved at 1 mg / ml in 0.9% NaCl solution. The product is diluted so that it can inhibit 80% of the anti-FXa or anti-FIIa activity present in the test article. The bead solution is made 1 mg / ml by diluting with a wash buffer of 20 mM Trismaleic acid, 150 mM NaCl, pH 7.35. The solution is agitated and 100 μl of the bead containing solution (1 mg / ml) is placed in an eppendorf tube. Add 500 μl of buffer. Centrifuge the tube at 12000 rpm for 5 minutes. After removing the supernatant, the pellet is dissolved in 500 μm buffer. After stirring, centrifuge for the second time and discard the supernatant again. Next, the product solution is the product / bead ratio expressed as 1 μg of product μg / avidin (Sigma Avidin Ref. A-9275, approximately 3 mg / ml solution depending on the batch). Are placed in contact with various solutions including beads such as having at 1, 0.1, 0.01 and 0.001. The mixture is then agitated and left standing for 1 hour before being centrifuged at 12000 rpm for 5 minutes. The supernatant is then removed and anti-FXa activity is analyzed to determine the concentration of product remaining in the supernatant. Anti-FXa or anti-FIIa activity has been demonstrated by Teien A. et al. N and Lie M.M. , Thrombosis Research, 1977, 10, 399-410. The results obtained are described in particular in Table 2.

  Thus, it can be seen that low molecular weight heparin is actually functionalized with biotin with a degree of biotinylation greater than 80% and can actually be neutralized with avidin.

  The biotinylated low molecular weight heparin according to the invention can be used for the preparation of a medicament. In particular, it can be used as an antithrombotic agent. Thus, according to another aspect of the present invention, an object of the present invention is an agent comprising a biotinylated low molecular weight heparin as defined above. These drugs are used in therapy, especially for venous thrombosis, arterial thrombosis (especially in the case of myocardial infarction or unstable angina), peripheral arterial thrombosis such as arterial disease of the lower extremities, cerebral artery thrombosis and stroke It finds use in therapy and prevention. These agents are also useful in the prevention and treatment of smooth muscle cell proliferation, angiogenesis, and as neuroprotective agents for atherosclerosis and arteriosclerosis.

  According to another aspect of the present invention, the present invention also relates to a method for the treatment of the above-mentioned pathological conditions, which method comprises administering an effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof to a patient. Administration. Accordingly, the use of biotinylated low molecular weight heparin as defined above for the treatment and prevention of the above-mentioned pathologies forms part of the present invention and the biotinylated low molecular weight heparin is used for the treatment of these pathologies or Use for the manufacture of a prophylactic agent also forms part of the present invention.

  According to another aspect of the present invention, the object of the present invention is to provide a biotinylated low molecular weight heparin according to the present invention or a pharmaceutically acceptable salt thereof and also at least one pharmaceutically acceptable as an active ingredient. A pharmaceutical composition comprising an inert excipient. The excipient is selected according to the desired dosage form and method of administration, eg, oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, transmucosal, topical or rectal route.

  In each dosage unit, the active ingredient is present in an amount suitable for the assumed daily dose in order to obtain the desired prophylactic or therapeutic effect. Each dosage unit may contain 20 to 150 mg, advantageously 40 to 100 mg of active ingredient. These doses of anticoagulant compound can be neutralized by intravenous, bolus or infusion with doses of 0.2 to 2 g of avidin or streptavidin.

  There may be special cases where higher or lower dosages are appropriate, and such dosages are outside the context of the invention. According to normal practice, the appropriate dosage for each patient is determined by the physician according to the method of administration and the weight and response of the patient.

  The compounds according to the invention can also be used in combination with one or more other active ingredients useful for the desired treatment, such as antithrombotic, anticoagulant or antiplatelet aggregating agents. .

  The object of the present invention is also a method of using avidin or streptavidin, characterized in that it allows neutralization of biotinylated low molecular weight heparin according to the present invention. Thus, avidin or streptavidin can be used for the preparation of a biotinylated low molecular weight heparin neutralizing agent according to the present invention.

Claims (18)

A biotinylated low molecular weight heparin composition, its configuration polysaccharide has a weight average molecular weight between 3000 7000 Da, constituted at least 60% of the polysaccharide covalently bound to the biotin derivative Te these reducing end odor And the covalently linked constituent polysaccharide is represented by the general formula (I):

[Where:
-I is equal to 0 or 1,
-R1 is represented by the formula (a) or (b):

{Wherein j and k are the same or different and are integers that can take any value from 1 to 10. } Chain,
Biot is the formula (c):

Represents the biotin group of
-PE represents a polysaccharide chain having the general structure of heparin's constituent polysaccharide;
-X represents H or _SO 3 Na,
-Y represents _SO 3 Na or COCH _3,
The wavy line represents a bond located either below or above the pyranose ring plane to which it is attached. ],
Or a pharmaceutically acceptable salt thereof, a biotinylated low molecular weight heparin composition. i is equal to or equal to 0, the biotinylated low molecular weight heparin composition according to claim 1. i is equal to 1, and R1 is of the formula (a) (wherein, j is equal to 5.), characterized in that represent the chain of biotinylated low molecular weight heparin composition according to claim 1. i is equal to 1, R1 is of the formula (b) (wherein, j and k are the same, equal to 5.), characterized in that represent the chain of biotinylated low molecular weight according to claim 1 Heparin composition. Configuration of at least 80% of the polysaccharide is characterized by having a covalent bond with biotin derivative of these reducing end, biotinylated low molecular weight heparin composition according to claim 1. Configuration of at least 90% of the polysaccharide is characterized by having a covalent bond with biotin derivative of these reducing end, according to claim 1 or biotinylated low molecular weight heparin composition according to claim 5. Biotinylated low molecular weight heparin composition according to any one of the preceding claims, characterized in that the low molecular weight heparin is selected from enoxaparin, ardeparin, bemiparin, parnaparin and tinzaparin . Low molecular weight heparin
- 20% 9% of its configuration polysaccharide has a weight average molecular weight of less than 2000 Da,
- from 5% to 20% of its configuration polysaccharide has a weight average molecular weight of greater than 8000 Da,
- 86% from 60% of its configuration polysaccharide has a weight average molecular weight between 2000 8000 Da,
- the ratio between the weight average molecular weight to number average molecular weight is between 1.3 to 1.6, and - the low molecular weight heparin has a weight average molecular weight of between 5500Da from 3 500,
The biotinylated low molecular weight heparin composition according to any one of claims 1 to 6 , wherein the composition is a biotinylated low molecular weight heparin composition. A method for preparing a biotinylated low molecular weight heparin composition as defined in any one of claims 1 to 8 , comprising the following steps:
a) performing a reductive amination of low molecular weight heparin at a temperature between 20 and 80 ° C. in the presence of an amine salt and a reducing agent;
b) then using an activating group-(R1) _i -Bio t , where R1, i and Biot are as defined in any one of claims 1 to 4 in an aqueous medium or Performing the acylation in the presence of a base in an organic medium;
A method comprising the steps of: The following steps:
a) performing a reductive amination on low molecular weight heparin at a temperature between 50 and 80 ° C. in the presence of ammonium halide and borohydride salts;
b) Subsequently, using an activated ester form of the-(R1) _i -Biot group, the activated ester form being selected from N-hydroxysuccinimide or 3-sulfo-N-hydroxysuccinimide derivatives). Performing the acylation in the presence of a base in the medium;
The method of claim 9 , comprising: A drug comprising the biotinylated low molecular weight heparin composition according to any one of claims 1 to 8 . As an active ingredient, a pharmaceutical composition, characterized in that the biotinylated low molecular weight heparin composition according Contact and further comprising at least one pharmaceutically acceptable excipient in any one of claims 1 to 8 . An antithrombotic agent comprising the biotinylated low molecular weight heparin composition according to any one of claims 1 to 8 . Venous thrombosis, arterial thrombotic disorders, end treetops arterial thrombosis, for the treatment and prevention of cerebral artery thrombosis or stroke, smooth muscle cell proliferation, for the prevention and treatment of angiogenesis, as well as atherosclerosis and arteriosclerosis as neuroprotective agents for medicament according to claim 13. The drug according to claim 14, wherein the arterial thrombotic disorder is myocardial infarction or unstable angina. The drug according to claim 14, wherein the peripheral arterial thrombosis is a lower limb arterial disease. A method for neutralizing a biotinylated low molecular weight heparin composition according to any one of claims 1 to 8 using avidin or streptavidin . Use of avidin or streptavidin for the preparation of a neutralizing agent for a biotinylated low molecular weight heparin composition according to any one of claims 1-8 .

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